Over 90% of diabetics fall into the category of Type 2 diabetes and while the primary factors causing this disease are unknown it is clear that insulin resistance plays a major role in its development. Recent 13C NMR studies have demonstrated that abnormalities in liver and muscle glycogen metabolism are the major factors responsible for insulin resistance in patients with Type 2 diabetes. The overall goal of this proposal is the application of novel 13C and 31P NMR spectroscopic techniques in conjunction with state-of-the-art GC-MS techniques to investigate the cellular mechanisms of insulin resistance in these tissues. This approach has major advantages over existing techniques currently being used for clinical investigation in that it is I) noninvasive, ii) involves no ionizing radiation and iii) repeated measurements of biochemical metabolites (both isotopically labeled and unlabeled) in plasma and tissue can be performed which then yield localized metabolic flux rates and information regarding rate controlling steps of glucose metabolism. During the tenure of this grant they plan to 1) Identify the rate controlling step for insulin stimulated glucose metabolism in skeletal muscle in normal and insulin resistant individuals using a novel 13C NMR method to assess intracellular glucose concentrations, 2) Examine the mechanism of FFA induced insulin resistance on glucose transport activity and insulin signaling using a novel tandem mass spectrometry method to identify serine phosphorylation of IRS-1, 3) Examine the regulation of uncoupling protein3 (UCP3) expression and activity in human skeletal muscle using a novel 13C/31P method, 4) Identify the allosteric regulators of hepatic glucose production in normal and diabetic individuals, and 5) Examine the impact of FFA on the ability of insulin and glucose to suppress hepatic glucose production. It is anticipated that the results of these studies will yield new and important insights into the pathogenesis of insulin resistance in patients with type 2 diabetes and further elucidate the role of FFA in this process. This information will enable the rational development of novel therapeutic agents to prevent or reverse this pathologic condition.